Methods for extracting platelets and compositions obtained therefrom

ABSTRACT

This invention provides methods for extracting platelets, compositions obtained therefore, and methods for using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/682,782,filed Apr. 13, 2010, which is a §371 U.S. National Stage entry from PCTPatent Application No. PCT/US08/80053, filed Oct. 15, 2008, which claimsthe priority benefit of U.S. Provisional Application No. 60/980,159,filed Oct. 15, 2007, which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under contract numberHL007171 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for extracting platelets,compositions obtained therefrom, and methods for using the same.

BACKGROUND OF THE INVENTION

New blood vessel formation, or angiogenesis, is requirement for therepair and regeneration of damaged body tissues. This process requiresthe coordinated activity of multiple cell types including endothelialcells, smooth muscle and other mesenchymal cells, as well as cells ofthe monocyte/macrophage lineage. A great amount of work has been putinto identifying growth factors that can stimulate angiogenesis fortherapeutic purposes. Much attention has been focused on the moleculeVEGF (vascular endothelial growth factor) because of its central role inthe process.

VEGF is believed to be a very specific stimulator of endothelial cellsthat line the interior of blood vessels. Augmentation of motility,mitogenesis, and differentiation of endothelial cells can bedemonstrated in response to VEGF readily in cell culture. In addition,it has been shown that animals lacking the gene for VEGF die early indevelopment from a failure of blood vessel formation. Thus, it isbelieved that VEGF could be used for therapeutic purposes to stimulateangiogenesis, for example, in treatment of heart disease and peripheralvascular insufficiency. However, it has been shown that in someinstances delivery of VEGF to ischemic tissue leads to the formation ofvery weak, leaky blood vessels lacking the support needed from the othercell types. In these instances, VEGF alone was not able to stimulate theformation of entire mature, therapeutically valuable, blood vessels.Without being bound by any theory, it is believed that a single growthfactor alone is not sufficient to stimulate the coordinated activity ofall of the necessary cell types to bring about angiogenesis fortherapeutic purposes, e.g., in wound healing.

Wound healing is a complex cascade of cellular and biochemical eventswhich lead to wound closure and repair of tissues. Conventionally, threesuccessive phases are classically distinguished in the wound healingprocess: (1) the inflammatory phase, which corresponds to increasedvascular permeability and migration of leukocytes and macrophages; (2)the proliferative phase, characterized by, among others, fibroblastproliferation and collagen synthesis resulting in granulation tissueformation; and (3) the remodeling phase where collagen and granulationtissue rearrangements result in scar resorption.

One of the first events which usually occurs in a wound is bloodextravasation that results in platelet aggregation and impregnation ofthe wound with platelet and serum constituents. Among these constituentsare polypeptide growth factors, which are known to play a major role intissue regeneration. It has been shown that platelet a granules, whichare released by aggregated platelets, are one of the richestphysiological source of platelet-derived growth factor (PDGF) andtransforming growth factor-β (TGF-β), while serum contains high amountsof insulin-like growth factor I (IGF-I), IGF-II and their bindingproteins (IGF-BPs).

PDGFs include PDGF, platelet derived angiogenesis factor (PDAF), TGF-βand platelet factor-4 (PF-4), which is believed to be a chemoattractantfor neutrophils. PDGF is a mitogen and chemoattractant for fibroblastsand smooth muscle cells and is a stimulator of protein synthesis incells of mesenchymal origin, including fibroblasts and smooth musclecells. PDGF is also a nonmitogenic chemoattractant for endothelialcells.

TGF-β is a chemoattractant for macrophages and monocytes. Depending onthe presence or absence of other growth factors, TGF-β increases thetensile strength of healing dermal wounds. TGF-β also inhibitsendothelial cell mitosis, and stimulates collagen and glycosaminoglycansynthesis by fibroblasts.

Other growth factors, such as epidermal growth factor (EGF), TGF-α, andheparin binding growth factors (HBGFs) such as VEGF and fibroblastgrowth factor (FGF) and osteogenin, are also believed to be involved inwound healing. Epidermal growth factor, which is found in gastricsecretions and saliva, and TGF-α, which is made by both normal andtransformed cells, are structurally related and may recognize the samereceptors that mediate cell proliferation on epithelial cells. It isbelieved that both factors are involved in acceleratingre-epithelialization of skin wounds.

The in vivo mode of action of these growth factors involveschemoattraction at the wound site, cell proliferation, and collagensynthesis. One very-interesting feature of these products is that it hasbeen shown that some of them, for example, PDGF and IGFs, worksynergistically in stimulating wound repair.

Growth factors are, therefore, potentially useful for specificallypromoting wound healing and tissue repair. The addition of exogenousgrowth factors to a wound has been shown to increase the rate at whichthe wound is closed, the number of cells in the healing area, the growthof blood vessels, the total rate of deposition of collagen, and strengthof the scar.

Platelets are fragments of cells that circulate in the blood andparticipate in the early phases of wound healing by binding to damagedtissue and releasing a large variety of factors that begin and sustainthe healing process. Numerous factors, including VEGF, are known to bepresent in platelets that have a role in blood vessel formation makingthem a good source to look for therapeutically valuable mixtures. Anumber of studies have been done to show that isolates of growth factorsfrom platelets can enhance the healing of dermal wounds. In addition,surgeons in a number of areas routinely harvest and concentrateplatelets from the blood of patients (PRP, platelet-rich plasma) as asource of growth factors to be placed into specific areas of the body inneed of tissue regeneration.

Platelet-derived wound healing formulae (PDWHF) are known. For example,a platelet extract, which is in the form of a salve or ointment fortopical application, has been used by others in an attempt to facilitatewound healing. Unfortunately, most conventional PDWHFs are obtained bycentrifuging whole blood to obtain a plasma rich in platelets but freeof red blood cells and treating the platelet rich plasma with thrombinto stimulate the production of a releasate, which can be combined withcollagen. These PDWHFs typically have a limited concentration of growthfactors with high concentration of albumin (e.g., 9.9%). Accordingly,clinical utility of these conventional PDWHFs are limited. In addition,these PDWHFs typically contain essentially non-mature growth factors dueto the use of thrombin in obtaining PDWHFs. Furthermore, conventionalPDWHF production is relatively expensive, thereby making thecost-benefit ratio of conventional PDWHFs questionable.

Revised sentence: Unfortunately, most conventional PDWHFs are obtainedby centrifuging whole blood to obtain a plasma rich in platelets butfree of red blood cells and treating the platelet rich plasma them withthrombin to stimulate the production of a releasate, which can becombined with collagen

Autologous human platelet derived wound healing formula, made ofthrombin activated platelet a granules, has also been shown to inducethe healing of chronic ulcers, thus making growth factor extracts anadvantageous alternative to the use of recombinant growth factors.

Accordingly, there is a continuing need for better wound healingcompositions and methods for obtaining such compositions.

SUMMARY OF THE INVENTION

The present invention provides various platelet extract compositionsthat are useful in treating a wound, and promoting angiogenesis and/ortissue regeneration; methods and processes for producing the same fromplatelets; and methods for using the same.

Some aspects of the invention provide compositions comprising a growthfactor, an angiogenesis activity, a tissue regenerative activity, or acombination thereof. Some compositions of the invention are produced bya process comprising:

-   -   disrupting pelletized platelets in an extraction solution to        produce an insoluble material portion and the extraction        solution comprising a growth factor; and    -   removing insoluble materials and proteins having molecular        weight of 2 kD or less from said extraction solution to produce        a composition comprising a growth factor.

The extraction solution can comprise ethanol, water, HCl, urea,guanidine, Phosphate buffer, NaCl, CaCl2, Arginine, Tris buffer, or amixture thereof. However, it should be appreciated that other solventsand/or compounds can also be present in the extraction solutiondepending on particular needs.

In some embodiments, methods for disrupting pelletized plateletscomprise sonication, homogenization, nitrogen cavitation, a Frenchpressure cell, or a combination thereof.

Yet in other embodiments, the step of removing insoluble materials andproteins comprises:

-   -   (i) filtering said extraction solution using an ultrafiltration        process to remove insoluble materials and proteins having        molecular weight of 2 kD or less; or    -   (ii) removing insoluble materials from said extraction solution;        and        -   (a) dialyzing said extraction solution against an acidic            solution using a semipermeable membrane having 2 kD or less            MWCO; or        -   (b) filtering said extraction solution using an            ultrafiltration process to remove proteins having molecular            weight of 2 kD or less.            Within these embodiments, in some instances, the step of            dialyzing comprises using a semipermeable membrane having 8            kD or less MWCO.

Still in other embodiments, the process further comprises subjecting theextraction solution to an ultrafiltration process to remove anymicroorganisms that maybe present. Within these embodiments, in someinstances the process further comprises lyophilizing the extractionsolution to produce a solid composition comprising the growth factor. Insome cases, the process further comprises dissolving the solidcomposition in a solution to produce a fluid composition comprising thegrowth factor.

In other embodiments, compositions of the invention comprise VEGF,TGF-β1, TGF-β2, PDGF-BB, EGF, or a mixture thereof.

Other aspects of the invention provide a wound healing compositioncomprising therapeutically effective amount of a composition of theinvention and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier comprises abandage. In other embodiments, the pharmaceutically acceptable carriercomprises a pharmaceutically acceptable polymer.

Still other aspects of the invention provide methods for regenerating atissue or treating a wound comprising contacting a desired site fortissue regeneration or the wound with an angiogenic compositioncomprising a composition of the invention.

In some embodiments, the desired site for tissue regeneration comprisesa burn site, a graft donor site, an oral and maxillofacial surgery site,a mucogingival surgery site, a perioplastic surgery site, an oral mucosasite, or a combination thereof.

Yet other aspects of the invention provide methods for reducing the riskof microbial infection in a wound comprising treating the wound with awound treatment composition comprising a composition of the invention.

Some aspects of the invention provide processes for producing a plateletextract having an angiogenic activity. Such methods typically comprise:

-   -   disrupting pelletized platelets in an extraction solution to        produce an insoluble material portion and the extraction        solution comprising a growth factor; and    -   removing insoluble materials and proteins having molecular        weight of 2 kD or less from the extraction solution to produce a        platelet extract having an angiogenic activity.

In some embodiments, methods for disrupting pelletized plateletscomprise sonication, homogenization, nitrogen cavitation, a Frenchpressure cell, or a combination thereof.

Yet in other embodiments, the step of removing insoluble materials andproteins comprises:

-   -   (i) filtering the extraction solution using an ultrafiltration        process to remove insoluble materials and proteins having        molecular weight of 2 kD or less; or    -   (ii) removing insoluble materials from the extraction solution;        and        -   (a) dialyzing the extraction solution against an acidic            solution using a semipermeable membrane having 2 kD or less            MWCO; or        -   (b) filtering the extraction solution using an            ultrafiltration process to remove proteins having molecular            weight of 2 kD or less.            Within these embodiments, the step of dialyzing comprises            using a semipermeable membrane having 8 kD or less MWCO.

Still in other embodiments, the process further comprises subjecting theplatelet extract to an ultrafiltration process to remove anymicroorganisms that maybe present.

Yet in other embodiments, some processes of the invention furthercomprise lyophilizing the platelet extract to produce a solid plateletextract. Within these embodiments, in some instances processes of theinvention further comprise dissolving the solid platelet extract in asolution to produce a platelet extract solution.

Some applications for compositions of the invention include treatingburns of all kinds, and treating the wound that is created from thegraft site when skin is taken from one site to be grafted onto a burn;all dental applications, in particular oral and maxillofacialapplications including, without limitation, bone grafting, guided tissueregeneration, involving, without limitation, the maxillo (upper jaw) ormandible (lower jaw), tissue regeneration, mucogingival surgery,perioplastic surgery, or other regenerative tissues including, withoutlimitation, the oral mucosa. Compositions of the invention can also beused in ENT applications; orthopedic wound healing applications, inparticular joint replacement and tendon-bone graft; in surgicalclosures, in particular plastic surgery for scar reduction; treatingbattlefield wounds; in cardiovascular applications (in particularmyocardial infarction); and chronic non-healing skin ulcers (e.g.,especially prevalent in diabetics).

Other uses include treating external wounds. In addition to externalwounds discussed above, exemplary external wounds that can be treated bycompositions and processes of the invention include, but are not limitedto, cuts (including surgical cuts or incisions), diabetic neuropathiculcers, pressure ulcers (e.g., bed sores), regions of gingival recessionundergoing surgical reconstruction, and any other external injuries thatbreak tissues or skin.

Compositions and processes of the invention can also be used to treatinternal wounds. For example, compositions and processes of theinvention can be used to treat cardiovascular disease, peripheralvascular insufficiency, spinal cord injuries, gastrointestinal injuries(e.g., wounds resulting from ulcers), hepatic injuries, and any otherinjuries that require tissue regeneration and/or angiogenesis.

In one particular embodiment, compositions and processes of theinvention are used to facilitate healing dental or oral wounds. Forexample, compositions and processes of the invention can be used tofacilitate wounds from oral surgery, tooth extraction, toothreplacement, tooth implants, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are magnified views of the cross-section of a Matrigelplug without and with the composition of the invention, respectively.

FIG. 2 is a graph showing counts of tubes/field within Matrigel plugsplaced subcutaneously in mice for two units of platelets obtained fromdifferent patients processed by sonication in physiologic media followedby dialysis against 10 mM HCl followed by lyophilization andresolubilization in concentrated urea and then dialysis again against 10mM HCl.

FIG. 3 is a bar graph showing the corresponding counts ofcapillaries/field of FIG. 2.

FIG. 4 is a picture showing the result of wound healing in mice with andwithout the composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “pelletized platelets” refers to platelets that have beenconcentrated (e.g., by centrifugation or filtration) such that the bulk(typically 90%, often 95%, and more often 98%) of the natural media(e.g., serum, plasma, anticoagulant solution) in which the platelets arenaturally found in have been removed.

When referring to platelets, the term “disrupted” means membranes ofplatelet cells and granules have been destroyed such that the contentsare released.

“Low molecular weight proteins” refers to proteins having a molecularweight of 10 kD or less, typically 8 kD or less, and often 2 kD or less.

“Ultrafiltration” refers to a filtration process that removesmicroorganisms such as bacteria and/or virus. Typically, ultrafiltrationis a membrane filtration in which hydrostatic pressure forces a liquidagainst a semipermeable membrane. Suspended solids, solutes of highmolecular weight, and microorganisms are retained, while water and lowmolecular weight solutes pass through the membrane. This separationprocess is used in industry and research for purifying and concentratingmacromolecular (10³-10⁶ Da) solutions, especially protein solutions.

A “pharmaceutically acceptable carrier” refers to any material or meansthat is used to deliver, transport, release, apply, or otherwise place aproduct or a composition of the invention to a desired or affected site.Exemplary pharmaceutically acceptable carriers include, but are notlimited to, polymers (including binders, slow release polymerformulations, polymers used as an artificial skin, and otherpharmaceutically acceptable polymers), bandages (including fabricbandages, polymeric bandages, and other solid materials that are used totreat wounds), solutions (such as those used in elixir, solutions usedin intravenous administrations, and other pharmaceutically acceptableliquids), as well as all known materials that are used in treatingwounds.

“Pharmaceutically acceptable excipient” refers to an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

“Treating” or “treatment” of a wound includes: (1) accelerating woundhealing, i.e., causing the wound to heal faster relative to an untreatedwound; (2) reducing the risk of microbial infection of a wound relativeto an untreated wound; and/or (3) reducing or relieving the painassociated with the wound.

“Wound” refers to any injury or condition that requires tissuegeneration or regeneration and is dependent on the formation of newblood vessels, or angiogenesis. A wound can be internal or external.Exemplary wounds include, but are not limited to, wound of the skin suchas a cut or ulcer, gingival recession around the teeth, regeneration ofnerve cells in the brain or spinal cord, and the condition of ischemia(diminished blood flow) to muscle in the heart or lower extremities.Generally, successful wound healing includes at least a partialrestoration of tissue function.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology, (2d ed. 1994); The Cambridge Dictionary of Science andTechnology, (Walker ed., 1988); and Hale & Marham, The Harper CollinsDictionary of Biology, (1991).

Products and Processes of the Invention

Platelets, or thrombocytes, are small cytoplasmic bodies derived fromcells. They circulate in the blood of mammals and are involved inhemostasis leading to the formation of blood clots. Like red bloodcells, platelets have no nucleus. Platelets contain alpha and densegranules. Activated platelets excrete the contents of these granulesinto their canalicular systems and into surrounding blood. There are twotypes of granules: (i) dense granules, which contain inter alia ADP orATP, calcium and serotonin; and (ii) α-granules, which contain interalia platelet factor 4, PDGF, fibronectin, B-thromboglobulin, vonWillebrand's factor (vWF), fibrinogen, and coagulation factors V andXIII).

The inner surface of blood vessels is lined with a thin layer ofendothelial cells, that in normal hemostasis, acts to inhibit plateletactivation with the production of endothelial-ADPase, noradrenaline, andPGI₂. Endothelial-ADPase clears away ADP, a platelet activator, fromplatelet surface receptors. Under the layer of endothelial cells is alayer of collagen. When the endothelial layer is injured, the collagenis exposed.

Endothelial cells produce vWF protein, a cell adhesion ligand that helpsendothelial cells adhere to collagen in the basement membrane. Undertypical physiological conditions, neither collagen nor vWF pass into thebloodstream. When endothelial damage occurs, platelets come into contactwith exposed collagen and vWF, and the inhibitors the endotheliumnormally secretes are reduced. Upon contact with collagen, the plateletsare activated. They are also activated by thrombin (primarily throughPAR-1), ADP receptors (P2Y1 and P2Y12) expressed on platelets. Plateletactivation results in the scramblase-mediated transport of negativelycharged phospholipids to the platelet surface. These phospholipidsprovide a catalytic surface (with the charge provided byphosphatidylserine and phosphatidylethanolamine) for the tenase andprothrombinase complexes.

Platelets participate in the early phases of wound healing by binding todamaged tissue and releasing a large variety of factors that begin andsustain the healing process. Numerous factors, including VEGF, are knownto be present in platelets that have a role in blood vessel formation.Isolates of growth factors from platelets have been shown to enhance thehealing of wounds.

Unfortunately, most conventional platelet-derived wound healing formulae(PDWHFs) are obtained by centrifuging whole blood to obtain a plasmarich in platelets but free of red blood cells and treating the plateletrich plasma with thrombin to stimulate the production of a releasate,which can be combined with collagen. These PDWHFs typically have alimited concentration of growth factors with high concentration ofalbumin (e.g., 9.9%) and other serum contents. Accordingly, clinicalutility of these conventional PDWHFs are limited. In addition, thesePDWHFs typically contain essentially non-mature growth factors due tothe use of thrombin in obtaining PDWHFs.

In contrast to compositions obtained from conventional treatment orextraction of platelets, compositions of the present invention have asignificantly higher angiogenesis activity and growth activity.Furthermore, surprisingly and unexpectedly, the present inventors havefound that using pelletized platelets provide a significantly improvedgrowth factor activity and angiogenesis activity. More significantly,use of pelletized platelets significantly reduces the amount ofundesired material in the composition, such as albumin, serum, plasma,etc.

Some aspects of the invention provide compositions that comprise agrowth factor, platelet extract mixtures having an angiogenic activity,as well as other compositions that are useful in promoting angiogenesisor tissue regeneration, treating a wound, or a combination thereof.Various compositions of the invention are obtained by processes of theinvention. Typically, processes of the invention comprise disruptingpelletized platelets in an extraction solution such that the contents ofthe platelets and/or the granules are released into the extractionsolution. Generally the extraction solution comprises one or more of thefollowing components, water (typically deionized water), HCl, urea,guanidine, Phosphate buffer, NaCl, CaCl₂, Arginine, Tris buffer, or amixture thereof. When pelletized platelets are placed in the solutiondescribed above, in certain instances at least a portion of plateletsand granules are disrupted, thereby releasing contents of platelets andgranules into the solution without a need for any mechanical force. Insome embodiments, pelletized platelets are further disrupted, e.g., byan external or mechanical force, to cause platelets and granules torelease their contents into the extraction solution. A variety ofdisruption methods are known to one skilled in the art, and all suchmethods are within the scope of the present methods. In some particularembodiments, platelets and granules are further disrupted by sonication,homogenization, nitrogen cavitation, a French pressure cell, or acombination thereof.

In some embodiments, the solution comprises a chaotropic agent. Withoutbeing bound by any theory, it is believed that the presence of thechaotropic agent has the benefit of inactivating or destroying thelysosomal proteases that are present in the platelets, which couldotherwise lead to the degradation of the desired product in the absenceof the chaotropic agent. Deionized water in certain instances can act asthe chaotropic agent. Other exemplary chaotropic agents include urea,guanidine, as well as those known to one skilled in the art. In someinstances, the chaotropic agent is urea. In such instances, theconcentration of urea used is typically about 4 M or higher, often about6 M or higher, and more often about 8 M.

Processes of the invention can further include removing at least aportion of insoluble materials from the extraction solution. Theinsoluble materials are produced by disruption of the pelletizedplatelets and granules and comprises membrane fragments and/or otherinsoluble higher molecular weight proteins. Any of the known separationmethods can be used to remove insoluble materials from the solution. Forexample, insoluble materials can be separated by centrifugation followedby collection of the supernatant, or the insoluble materials can beremoved simply by filtration.

Processes of the invention can further include removing proteins havingmolecular weight of about 2 kD or less. Such proteins can be removedalong with the insoluble materials, for example, using anultrafiltration process. Alternatively, the insoluble materials can beremoved from the extraction solution prior to removing proteins havingmolecular weight of 2 kD or less. For example, after removing theinsoluble materials the resulting extraction solution can be dialyzedusing a semipermeable membrane having 2 kD or less, often 4 kD or less,and more often 10 kD or less MWCO. Without being bound by any theory, itis believed that dialysis removes various proteins that may interferewith the tissue regeneration, tissue growth, or angiogenic activity ofthe platelet extract. While it is possible that some of the desiredmaterials (e.g., materials having angiogenic activity, growth factors,etc.) may also be removed in the process of removing low molecularweight proteins, the net result is a composition having an increasedtissue growth, tissue regeneration, and/or angiogenic activity. It isbelieved that in some instances the increased activity is a result ofremoving materials that may have inhibitory activity or simply thatmaterials that bind to growth factors or angiogenic materials areremoved from the extract, thus reducing the amount of antagonists. Ithas been found by the present inventor that dialysis against an acidicsolution, often a dilute acidic solution such as 10 mM HCl solution,provides platelet extracts with a significantly increased growth and/orangiogenic activities.

In some embodiments, processes of the invention also include subjectingthe extraction solution to an ultrafiltration process to remove anymicroorganisms, such as virus and/or bacteria, that maybe present. Whena relatively long term storage of the composition is desired, it isoften advantageous to store such a composition in a solid form, whichreduces the likely hood of contamination or increases the stability ofthe composition. A solid form of the compositions of the invention canbe obtained by any of the known processes, for example, bylyophilization. Lyophilization may also take place with the presence ofsucrose, trehalose or other compounds to maintain biologic activity ofthe proteins or other additive as is commonly practiced in the art oflyophilization. Platelet extract preparations or pharmaceuticalcompositions comprising the same can be frozen and subsequently thawedor lyophilized and reconstituted/re-dissolved, for example with anextraction solution as defined herein. Such reconstituted solution canbe used with or without dialysis against dilute acid or acidification.

Without being bound by any theory, platelet extract compositions of theinvention are believed to include various growth and/or angiogenesisfactors including, but not limited to VEGF, TGF-β1, TGF-β2, PDGF-BB,EGF, or a mixture thereof.

In some embodiments, compositions of the invention can include a smallamount of albumin content. Generally the amount of albumin in productsof the invention is about 1% or less, often about 0.7% or less, and moreoften about 0.4% or less. In general, compositions of the invention havea higher content of one or more wound healing or angiogenic substances(e.g., growth factors, fibronectin, thrombospondin, etc.) than plateletextract products obtained from any conventional process. Accordingly,compositions of the invention typically have a higher wound healing ortissue regeneration activity compared to platelet extract products ofconventional processes.

The source of platelets is not important as compositions of theinvention can be obtained from even what is known in the art as expiredhuman platelets. Typically, apheresed platelets maintain theirviablility for transfusion for about six days. After six days, theseapheresed platelets are considered expired because of the risk ofculturing infection causing bacteria.

In one particular embodiment of the invention, processes of theinvention provide a mixture of proteins or materials that are known toaid in wound healing or tissue regeneration. In this particularembodiment, processes of the invention include centrifuging plateletsand removing the serum supernatant to obtain pelletized platelets,resuspending pelletized platelets in a solution such as, but not limitedto, PBS or 10 mM HCl solution or a solution comprising a chaotropicagent. The resuspended materials are subjected to conditions to destroythe membranes, for example, lysing by sonication to destroy themembranes of the platelets and granules. In some cases, the resultingsupernatant after lysing and centrifugation is acidified to about pH 4or less, typically about pH 3 or less, and more often about pH 2 orless. In one particular embodiment, the supernatant is acidified toabout pH 2.

As stated above, a chaotropic agent, such as urea, can be included inthe initial extraction solution. Without being bound by any theory, itis believed that the presence of the chaotropic agent has the benefit ofinactivating or destroying the lysosomal proteases that are present inthe platelets, which could otherwise lead to the degradation of theproduct in the absence of the chaotropic agent.

The acidified mixture is dialyzed against 10 mM HCl solution using a 7-8kD membrane. The resulting material is lyophilized for storage andresolubilized in a chaotropic agent prior to its use. Exemplarychaotropic agents include urea, guanidine, as well as those known to oneskilled in the art. In one particular embodiment, the chaotropic agentis urea. In such embodiment the concentration of urea is typically about4 M or higher, often about 6 M or higher, and more often about 8 M. Thereconstituted solution is then dialyzed against 10 mM HCl to remove thechaotropic agent (e.g., urea). Without being bound by any theory, it isbelieved that acidification and chaotropic agent (e.g., urea)solubilization activates latent forms of TGF-β super family members suchas TGF-βs and BMPs. Another method of activating TGB-β super familymembers includes heat treatment.

Compared to platelet extract products of conventional processes,products of the invention have superior therapeutic utility in thetreatment of conditions known to result from insufficient angiogenesissuch as impaired wound healing, and myocardial and peripheral vascularischemia.

Other methods for isolating or extracting materials from plateletsinclude, but are not limited to, subcellular fractionation of theplatelets by a process such as nitrogen cavitation or the use of aFrench pressure cell. In some instances, density centrifugation on thelysis product yields a system of bands that allow one to isolate theα-granules which are the organelles that comprise the factors that showwound healing properties. In some instances, the isolated α-granules arefurther subjected to processes of the invention, e.g., chaotropic agent(e.g., urea) treatment.

Other aspects of the invention include compositions comprising atherapeutically effective amount of a product of the invention and apharmaceutically acceptable carrier, and methods for using the productor the composition of the invention.

Utility

The dynamic process of wound healing is a well regulated sequence ofevents which, under normal circumstances, results in the successfulrepair of injured tissues. First, a cutaneous wound that cuts throughthe epidermis and dermis (full thickness), is accompanied by bloodvessel rupture. Rapidly, clot formation occurs providing a provisionalmatrix to cover the wound. The clot is one of the key components becauseit provides mechanical closure with fibrin and other matrix proteins,and it is the initial source of cytokines, growth factors andchemotactic agents released by platelet degranulation. This cocktailinitiates the process of wound healing. Next, neutrophils move into theinterstitum at the site of injury in response to bacterial products andother chemotactic agents. This is followed by macrophages that releasechemical signals to attract fibroblasts. The resident and infiltratingfibroblasts secrete cytokines such as PDFG-BB and bFGF and begin todeposit a new extracellular matrix that will be an essential componentof the scar tissue. Meanwhile, the process of reepithelialization beginson the borders of the wound where keratinocytes of the basal layerdisplay new integrins to attach to a provisional matrix. The epidermalmigration continues until a monolayer of keratinocytes covers the wound.Several known growth factors intervene in the reepithelialization of theskin (e.g., EGF, TGFa and KGF 1 and 2).

In the underlying dermis, the process of neovascularization isestablished in response to severed vessels and angiogenic factorsproduced locally. The role of the microvasculature in wound healing isessential for the repair to take place. After the interruption in thecontinuity of the microvasculature, endothelial cells need to dissolvetheir cell-cell attachments, migrate outside the vessel into theextracellular matrix, undergo mitosis and finally reassociate in anorderly manner to form a network of capillaries necessary for thehealing to proceed. It appears that VEGF secreted acutely by thekeratinocytes is responsible in great part for the angiogenic response.Other angiogenic factors like basic fibroblast growth factor (bFGF) andtransforming growth factor b (TGFb) are also present.

Normal healing involves proliferation, migration, matrix synthesis andangiogenesis. An impairment at any of these complex phases often willlead to complications in wound healing. In diseases of impairedneovascularization, such as diabetes, dermal wound healing is severelycompromised. This often leads to nonhealing wounds and, ultimately,amputation. Compositions of the invention stimulate and/or augmentangiogenesis and can be of great value in diabetes and other clinicalsituations where healing is impaired.

Compositions of the invention promote wound healing or tissueregeneration and can be used to treat external or internal wounds.Furthermore, compositions of the invention can be used topically orsystemically. Generally any type of external or internal wound can betreated with compositions of the invention to promote or facilitatewound healing. For example, compositions of the invention can be used intreating cutaneous and surface wounds. Exemplary uses include treatingsimple cuts and scratches, surgical wounds to decrease scar tissueformation (e.g., in the form of a bandage), treating dental and internalwounds such as in surgical applications, dental graft healing,battlefield wound treatment, treatment of myocardial infarction andother internal ischemic events. Compositions of the invention can alsobe used in dental and oral and maxillofacial applications, including,without limitation, bone grafting, guided tissue regeneration,involving, without limitation, the maxillo (upper jaw) or mandible(lower jaw), tissue regeneration, mucogingival surgery, perioplasticsurgery, and other regenerative products including, without limitation,the oral mucosa. Other applications for the compositions of theinvention include orthopedic applications (such as in ligament to boneand tendon to bone attachments), joint replacement applications (e.g.,shoulders, knees, hip, etc.), treating diseased or damaged cells incardiovascular system, treating burns, mucosal cell regeneration (e.g.,in trachea, gynecology, proctology, etc.), and in general for healingvarious internal and external wounds.

Compositions of the invention can also be combined with other materials.For example, they can be combined with Laminin purified from placenta asa wound healant or scar minimizer. They can also be combined withBiobrane® or some other wound covering for burns and graft siteregeneration. Compositions of the invention can also be loaded onto acollagen sponge to enhance tendon to bone healing when sandwiched orplaced between the tendon and bone. Furthermore, compositions of theinvention in carboxymethylcellulose can be used as a wound regenerantsimilar to Regranex®. In some instances, compositions of the inventionin dilute acid, physiological buffer, or deionized water can be applieddirectly to a wound bed to accelerate wound healing. Compositions of theinvention can also be used as a nutrient supplement for animal serumfree cell culture or primary human cell cultures. Compositions of theinvention can also be used as therapeutic angiogenesis agents in theheart and lower extremities and as osteogenic agents suitable for boneregeneration.

Some particular uses for compositions of the invention include softtissue grafting procedures involving the oral mucosa including treatmentof gingival recession, increasing the amount of keratinized tissue, andincreasing vertical and/or horizontal gingival tissue thickness. Otheruses for compositions of the invention include promoting papillaregeneration, vestibuloplasty, and tissue regeneration around dentalimplants. Compositions of the invention can also be used in esophagusreconstruction, oral facial reconstruction, and to repair mucosa tissuelost due to trauma or diseases such as cancer and periodontal disease.

Other application for compositions of the invention is to stimulateperiodontal regeneration including the formation of cementum,periodontal ligament, and bone when applied to the surface of rootsurfaces of teeth. Compositions of the invention can also be used intympanoplasty, tonsillectomy, adenoidectomy, tumor resection and woundmanagement, plastics and reconstruction surgery including, free tissuetransfer (split thickness flaps), bone grafting, Z-Plasty (or softtissue scar reduction), and augmentation (e.g., as micronized injectableand large particle grafting material.

In some applications, compositions of the invention can be mixed with avarious polymers or membrane, for example, with a membrane composed ofamnion and chorion (such as those disclosed in U.S. patent applicationSer. No. 12/206,508, which is incorporated herein by reference in itsentirety), or with a simple collagen membrane. Compositions of theinvention can also be combined with an amnion or collagen membrane forthe treatment of chronic wounds.

In some instances, solid compositions of the invention (e.g., those thatare lyophilized) are hydrated at the time of application (e.g., duringsurgery) using saline. The reconstituted composition is typicallyapplied directly to the desired site, for example, to the flap prior tocoronally positioning over the exposed root surface of adjunct tooth inthe treatment of gingival recession. Compositions of the invention canalso be used to the debrided bone and cleaned root surface in thetreatment periodontal osseous defects and simulate periodontalregeneration.

As stated above, compositions of the invention can be lyophilized andhydrated at the time of use, e.g., during surgery, using saline. Suchreconstituted composition can be applied to a collagen membrane. In oneparticular embodiment, compositions of the invention is applied to acollagen membrane that is 1.5 mm thick, where the top layer of themembrane consists of amnion tissue with epithelia-layer removed, forexample, using methods disclosed in the above incorporated U.S. patentapplication Ser. No. 12/206,508. The hydrated membrane comprising thecomposition of the invention can be used as alternative to free gingivalgrafts to increase the zone of attached gingival tissue or to help grownew gingival tissue around the exposed surface of a dental implant.

Other applications for compositions of the invention include bonegrafting applications. For example, compositions of the invention can beused in dental bone grafting procedures including ridge augmentation,sinus elevation, facial reconstruction, extraction site grafting andperiodontal osseous defects, where the composition of the invention ismixed with autogenous bone, and or a bone substitute. Compositions ofthe invention can also be mixed with autogenous bone, and/or a bonesubstitute, and used in conjunction with a barrier membrane for use inguided bone regeneration.

In another particular embodiment, a solution composition of theinvention is lyophilized and hydrated at the time of surgery usingsaline, and mixed with particulate an organic bone mineral, such asthose with an average diameter range of from about 250 μm to about 420μm, and is used to treat periodontal osseous defects.

Compositions of the invention also have applications in spine, trauma,joint reconstruction, and craniofacial procedures, for example, wherecompositions of the invention are mixed with autogenous bone, and/or abone substitute. One of the objectives of spinal fusion is to eliminatethe excessive vertebral motion or spinal instability by fusing adjoiningvertebrae. In cases where the amount of autograft bone required exceedsavailability, surgeons mix in a bone substitute product usually called abone graft “extender” to have enough material for the procedure. As thenumber of “levels” (vertebras) fused increases, so does the difficultyof the procedure. There are two primary spinal fusion procedures. One isinterbody fusions, which use a device that is inserted into the discspace between the vertebrae. In this procedure, the inner portion of theinterbody device, which is usually hollow, is filled with bone graftmaterial. Another often used procedure is a posterolateralintertransverse process, which typically requires a large amount of bonegraft material (usually autograft) to be placed in the posterolateralportion of the spine with a goal of fusing the transverse processestogether. In both fusion procedures, spinal fixation devices such aspedicle screw systems, rods and plates are typically used to helpstabilize the joint. As stated, compositions of the invention can beused in various spine fusion procedures. Compositions of the inventionare also useful in intertranverse fusion process where natural bloodsupply is provided only by the de-corticated pedicles above and belowthe bone graft.

In fractures bone graft materials are used to treat both fresh fractures(result of a recent injury) and non-union fractures (a fresh fracturethat did not heal properly). In these procedures that require a bonegraft, the material is packed into the fracture site in order to fillthe void and heal the bone. In addition to the bone graft material, thefracture is typically stabilized using fracture fixation devices.Compositions of the invention mixed with a bone substitute can be usedin all fractures, grade A1-C3. In simple fractures such as distal radiusfactures and foot and ankle fusions, compositions of the invention canshorten recovery time. In non-healing fractures or factures where theblood supply is compromised, compositions of the invention can be aneffective alternative to autograft or can be used at the very least as abone graft extender.

Following diseases and/or surgical intervention human and other animaltissues heal by replacement with a fibrotic scar tissue. Such tissuedoes not generally possess the functional characteristics of theoriginal tissue. Various attempts have therefore been made to obtain ahealing response characterized by regeneration, i.e., the replacement oflost tissue with a newly generated tissue functionally andmorphologically similar to the original one. In defined clinicalsituations both natural healing responses and guided tissue regenerationprocedures present several limitations which decrease the extent andpredictability of the desired healing outcome. Clinical application ofthe concept of guided tissue regeneration in periodontics (treatment ofgum diseases), implantology (replacement of lost teeth by artificialbone anchored ones), orthopedics, plastic surgery, etc. has proven to behighly effective in selected conditions characterized by a particulartopographic configuration of the tissue to be regenerated. In mostsituations, however, results are less satisfactory both in terms ofamounts of obtained regenerated tissue and their predictability.Classical explanations of this phenomenon have centered the attentionupon limits arising from insufficient recruitment of specific progenitorcells.

In some aspects, the invention provides a pharmaceutical compositionadapted to the use in guided tissue regeneration. In some embodiments,products, compositions and methods of the invention increasemicrovascular blood flow, recruit progenitor cells, increasemicrovascular endothelium proliferation, and/or protect tissues fromischemia and reperfusion damages. Various local delivery systems can beused. Generally, delivery systems do not negatively affect tissue (e.g.,periodontal) regeneration. The delivery composition can be used incombination with or without physical barriers such as plastic orresorbable polymer films. Specific formulations and applicationtechnique are adapted to the different applications.

Compositions of the invention also reduce the risk of microbialinfections. Accordingly, some aspects of the invention provide methodsfor reducing the risk of a microbial (e.g., bacteria, yeast, and/orfungus) infection of wounds.

Polymers of various types, in combination with compositions of theinvention, are useful in the present invention and include those polymermaterials that are safe for use in the oral cavity and wounds of humanor a lower animal. Such polymers are known, including for examplepolymers and copolymers such as polylactic acid (“PLA”), polyglycolicacid (“PLG”), poly lactyl-co-glycolic acid (“PLGA”), polyaminoacids suchas polyaspartame, chitosan, collagen, polyalburrin, gelatin andhydrolyzed animal protein, polyvinyl pyrrolidone xanthan and other watersoluble gums, polyanhydride, and poly orthoesters.

In some embodiments, useful polymers include the copolymers comprisingmixtures of lactide and glycolide monomers. Lactide monomeric speciescan comprise from about 15% to about 85%, often from about 35% to about65% of the polymers, while glycolide monomeric species comprise fromabout 15% to about 85% of the polymer, often from about 35% to about 65%on a molar basis. However, it should be appreciated that the scope ofthe invention is not limited to any particular copolymer rangesdisclosed or exemplified herein. The range of copolymer contents canvary according to a particular application. Generally, the molecularweight of the copolymer typically lies in the range of from about 1000to about 120,000 (number average). These polymers are described indetail in U.S. Pat. No. 4,443,430, issued to Mattei, which isincorporated herein by reference in its entirety.

A feature of fluid gel or paste-like compositions containing certain ofsuch copolymers is their transformation into near solid phase in thepresence of aqueous fluid such as water, aqueous buffers, serum,crevicular fluid, or other body fluid. For example, when a sample ofsuch a gel is placed into a tube containing water or human serum, thecomposition becomes nearly solid in the receptor phase. Without beingbound by any theory, this is believed to be due to insolubility of thepoly(lactyl-co-glycolide) copolymer in water, and related aqueoussolvents such as may be present in wound or crevicular fluid. Thus, eventhough such fluid compositions can potentially be used advantageouslywhen desired from a syringe-like apparatus, they still offer theadvantages of solid devices at the treatment sites such as dentalapplications. Further, since such polymeric materials do undergo slowdegradation via hydrolysis, the product of the invention continues torelease in a sustained manner from such compositions and the compositiondoes not need to be surgically removed following tissue regeneration.

In some embodiments of dental applications, the product of the inventiongenerally comprises from about 1% to about 90%, often from about 10% toabout 70%, of the compositions/devices useful for the methods of theinvention. It should be appreciated, however, that the scope of theinvention is not limited to any particular ranges given here and in theExamples. One skilled in the art can readily determine the appropriateamounts and ranges of the product of the invention depending on theparticular composition, device, and/or wound to be treated.

Administration and Composition

In treating wounds, the products of the invention can be utilized incompositions such as tablets, capsules or elixirs for oraladministration, suppositories for rectal administration, sterilesolutions or suspensions for parenteral or intramuscular administrationand the like.

The pharmaceutical compositions of the invention can be used in the formof a pharmaceutical preparation, for example, in solid, semi-solid orliquid form which comprises a product of the invention, as an activeingredient, in admixture with an organic or inorganic carrier orexcipient suitable for external, enteral or parenteral applications. Theactive ingredient can be compounded, for example, with the usualnon-toxic pharmaceutically acceptable carriers for tablets, pellets,capsules, suppositories, solutions, emulsions, suspensions, and anyother form suitable for use. The carriers which can be used are water,glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea and other carriers suitable for use in manufacturingpreparations, in solid, semisolid or liquid form and in additionauxiliary, stabilizing, thickening and coloring agents and perfumes canbe used. The active product (e.g., an agent that mediates angiogenicand/or wound healing capability) is included in the pharmaceuticalcomposition in an amount sufficient to produce the desired effect uponthe process or condition (e.g., regulation of neovascularization) of thedisease.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier (e.g., conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums) andother pharmaceutical diluents (e.g., water) to form a solidpreformulation composition containing a substantially homogeneousmixture of a product of the invention. When referring to thepreformulation compositions as substantially homogenous, it is meantthat the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms, in which the novel composition of the invention can beincorporated for administration orally or by injection, include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for reconstitution with water or other suitable vehiclesbefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, methyl cellulose or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); andartificial or natural colors and/or sweeteners.

For buccal administration, the composition may take the form of tabletsor lozenges formulated in conventional manners.

For topical administration, formulations may be made up with a productof the invention which may be combined in admixture with at least oneother ingredient constituting an acceptable carrier, diluent orexcipient in order to provide a composition, such as a cream, gel,solid, paste, salve, powder, lotion, liquid, aerosol treatment, or thelike, which is most suitable for topical application. Sterile distilledwater alone and simple cream, ointment and gel bases may be employed ascarriers of the active agents. Examples of bases and suspending vehiclesinclude Fattibase® (acrylic polymer resin base), Polybase® (polyethyleneglycol base) by Paddock Laboratories, Inc. Additional therapeutic agentsmay be added to the formulations as medically indicated, selected fromthe classes of: keratolytics, surfactants, counter-irritants,humectants, antiseptics, lubricants, astringents, wound additionalhealing agents, emulsifiers, wetting agents, additional adhesion/coatingprotectants, additional anti-inflammatory agents, vasoconstrictors,vasodilators, anticholinergics, corticosteroids (e.g., glucocorticoids)and anesthetics. Preservatives and buffers may also be added. Theformulation may be applied to a sterile dressing, biodegradable,absorbable patches or dressings for topical application, or to slowrelease implant systems with a high initial release decaying to slowrelease. When the compositions are administered to treat burns, they maybe in the form of an irrigant, preferably in combination with aphysiological saline solution. Compositions can also be in the form ofointments or suspensions, typically in combination with purifiedcollagen. The compositions may also be impregnated into transdermalpatches, plasters, and bandages. For additional topical compositionscontemplated for therapeutic administration, see U.S. Pat. No.5,662,904; U.S. Pat. No. 5,679,655; and U.S. Pat. No. 5,705,477, all ofwhich are incorporated herein by reference it their entirety.

The product of the invention can also be formulated for parenteraladministration by injection, which includes using conventionalcatheterization techniques or infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules, or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the activeingredients may be in powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Some aspects of the invention includes compositions comprising at leastone product of the invention together with at least one pharmaceuticallyacceptable carrier, and optionally other therapeutic and/or prophylacticingredients, such as antimicrobial compounds and analgesic compounds.

In general, the products of the invention are administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. Suitable dosageranges are typically 0.001 mg to 1 mg daily depending upon numerousfactors such as the severity of the wound to be treated, location of thewound, the age and relative health of the subject, the route and form ofadministration, the indication towards which the administration isdirected, and the preferences and experience of the medical practitionerinvolved. One of ordinary skill in the art of treating wounds will beable, without undue experimentation and in reliance upon personalknowledge and the disclosure of this application, to ascertain atherapeutically effective amount of the products of the invention for agiven wound treatment.

In some aspects of the invention, products of the invention areadministered as pharmaceutical formulations including those suitable fortopical, oral (including buccal and sub-lingual), rectal, nasal,pulmonary, vaginal, or parenteral (including intramuscular,intraarterial, intrathecal, subcutaneous and intravenous) administrationor in a form suitable for administration by inhalation or insufflation.Typically, the manner of administration is topical using a convenientpharmaceutical carrier such as bandages, artificial skin, polymers, andother wound dressing medium that are conventionally used. Additionally,synthetic products such as Dermagraft-TC, which is made from livinghuman cells and it is being used instead of cadaver skin, can be used.In addition to Dermagraft-TC, artificial burn dressings can be used,e.g., in treating burns. Exemplary artificial burn dressings includeIntegra Artificial Skin and BioBrane. Skin substitute BioBrane is anylon material that contains a gelatin that interacts with clottingfactors in the wound. It is believed that interaction causes thedressing to adhere better, forming a more durable protective layer.Unlike traditional bandages, artificial burn dressings also promotewound healing by interacting directly with body tissues.

Integra is a two-layered dressing. The top layer serves as a temporarysynthetic epidermis; the layer below serves as a foundation forre-growth of dermal tissue. The underlying layer comprises collagenfibers that act as a lattice through which the body can begin to aligncells to recreate its own dermal tissue.

Other substitute skin products can also be used with products of theinvention. For example, Apilgraf (Organogenesis Inc., Canada) is aliving “human skin equivalent” that can be used to treat wounds andulcers. Additionally, implantable human tissues (e.g., available fromLifeCell Corp.) can be used with products of the invention inreconstructive surgery and burn treatment.

A product of the invention, together with one or more conventionaladjuvants, carriers, or diluents, may be placed into the form ofpharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds or principles, and the unit dosage forms may containany suitable effective amount of the active ingredient commensurate withthe intended wound healing. The pharmaceutical compositions can beemployed as gels, ointments, solids (as a part of wound dressingmaterial such as bandages, artificial skins, casts, polymeric mixtures,semisolids, powders, and/or sustained release formulations), or liquidssuch as solutions, suspensions, emulsions, or elixirs; or in the form ofsuppositories for rectal or vaginal administration.

The products of the invention can be formulated in a wide variety ofmaterials for surgical placement forms. The pharmaceutical compositionsand dosage forms can comprise a product of the present invention as theactive component.

The pharmaceutically acceptable carriers can be either solid, gel, orliquid. Solid pharmaceutically acceptable carriers include, but are notlimited to, polymers (e.g., for placing the products of the invention ata desirable site such as spine, vertebrae, tooth, nerve cells,cardiovascular organs, digestive organs, or any other internal organs),bandages (including fabric and polymers), artificial skins, sutures,etc.

Other forms suitable for wound healing include gel or ointment formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, or solid form preparations that are intended to beconverted shortly before use to gel or liquid form preparations.Emulsions can be prepared in solutions, for example, in aqueouspropylene glycol solutions or may contain emulsifying agents, forexample, such as lecithin, sorbitan monooleate, or acacia. Aqueoussolutions can be prepared by dissolving the products of the invention inwater and adding suitable colorants, stabilizers, and thickening agents.Aqueous suspensions can be prepared by dispersing the finely dividedproducts of the invention in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well known suspending agents. Solidform preparations include solutions, suspensions, and emulsions, and maycontain, in addition to the active component, colorants, stabilizers,buffers, dispersants, thickeners, solubilizing agents, and the like.

The compounds of the invention also can be formulated for topicaladministration to the epidermis as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents.

The products of the invention can be formulated for administration assuppositories. A low melting wax, such as a mixture of fatty acidglycerides or cocoa butter is first melted and the active component isdispersed homogeneously, for example, by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and to solidify.

The compounds of the invention can be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

The compounds of the invention can also be formulated for nasaladministration. The solutions or suspensions are applied directly to thenasal cavity by conventional means, for example, with a dropper, pipetteor spray. The formulations may be provided in a single or multidoseform. In the latter case of a dropper or pipette, this can be achievedby the patient administering an appropriate, predetermined volume of thesolution or suspension. In the case of a spray, this can be achieved forexample by means of a metering atomizing spray pump.

The products of the invention also can be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The products will generally have a smallparticle size for example of the order of five (5) microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronization. The active ingredient is provided in a pressurizedpack with a suitable propellant such as a chlorofluorocarbon (CFC), forexample, dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, or carbon dioxide or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of drug may be controlled by a metered valve. Alternatively theactive ingredients may be provided in a form of a dry powder, forexample a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). The powder carrier will form agel in the nasal cavity. The powder composition may be presented in unitdose form for example in capsules or cartridges of e.g., gelatin orblister packs from which the powder may be administered by means of aninhaler.

When desired, formulations can be prepared for sustained or controlledrelease administration of the products of the invention. For example,the products of the invention can be formulated in transdermal orsubcutaneous delivery devices. These delivery systems are advantageouswhen sustained release of the product of the invention is desired ornecessary. Products in transdermal delivery systems are frequentlyattached to an skin-adhesive solid support. The compound of interest canalso be combined with a penetration enhancer, e.g., Azone(1-dodecylazacycloheptan-2-one). Sustained release delivery systems canbe inserted subcutaneously into the target organ or subdermal layer bysurgery or injection. In some instances, the subdermal implantsencapsulate the product of the invention in a lipid soluble membrane,e.g., silicone rubber, or a biodegradable polymer, e.g., polylacticacid.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES Example 1

The human platelet morphogen (HPM) material was made by transferring theunit of platelets into 50 mL centrifuge tubes (about 4-6 tubes) andcentrifuging at about 3500-4800 rpm to produce platelet pellets. Thesupernatant serum/media was removed using a pipette. A total of 60 mL ofcold 10 mM HCl solution, PBS, or 6-8 M urea with or without 5 mM CaCl₂was used to resuspend the platelet pellets which were then kept on iceduring the process of sonication using a Branson Sonifier 450 (BransonUltrasonics Corporation, Village of Lakewood, Ill.) with Duty cycle setto 20% and output control set to 2. Sonication was conducted for 4minutes with the probe tip placed in the liquid phase during theprocess. The resulting solutions were placed in a high G centrifuge for40 minutes to 1.5 hours. The resulting supernatants were collected anddialyzed in a 7-8 KD MWCO bag (5 1 1 exchanges lasting at least 6 hourseach) against cold 10 mM HCl solution to remove urea or buffer andsalts.

Bradford assays were done that yielded around 3-5 mg/mL (for the entire60 mL used) when lysis was done in dilute HCl or PBS and 7-8 mg/mL whendone in urea. All Matrigel plug assays were done with defined dosesbased on these concentrations. In some instances, this material waslyophilized and the weight of this product was measured yielding aweight approximately 10% greater than that determined by the Bradfordassay.

Example 2

A unit of expired platelets was obtained from the blood bank and theentire composition was distributed into 8×50 mL centrifuge tubes. Thesetubes were spun at 4000 rpm for 15 minutes and the supernatant wascarefully removed to yield a pellet of platelets at the bottom of eachtube. A 10 mL of solution composed of 6 M urea in 10 mM Tris pH 6.7buffer solution was placed in each tube and a clean pipette tip was usedto dissaggregate each pellet followed by brief vortexing (3-5 seconds)to further resuspend the pellet. Each tube was treated for two minutesby submerging a sonicator probe into the solution and activating thesonication for 30 seconds. The resulting solutions were loaded into 30mL centrifuge tubes and spun at 17000 rpm for 45 minutes. The resultingsupernatants were collected with a pipette and loaded into dialysis bagswith a 3.5 kD pore size and placed into one liter containers of chilled(about 4° C.) 10 mM HCl solution. Over a three day period thesesolutions were exchanged 4 times after a minimum of 8 hours with 1 literof fresh chilled 10 mM HCl solution. The solutions were then removedfrom the bags and stored in 15 mL centrifuge tubes in a refrigerator.

This resulted in a solution with a protein concentration by Bradfordassay of about 5.5 mg/mL. A total of 18.9 μL of this cold solution wasloaded into 1 mL of chilled, liquid Growth Factor Reduced Matrigel(Becton-Dickinson). This solution was injected in five ICR mice using a1 mL 27 g syringe under the skin of the lower abdomen in approximately200 μL aliquots to form a distinct plug in 5 ICR mice. Another 1 mL ofchilled liquid GFR Matrigel was loaded with 37 μL of 10 mM HCl solutionand injected in another 5 ICR mice under the skin of the lower abdomenin approximately 200 μL aliquots to form a distinct plug in each. Thisserved as the control group. After seven days the animals weresacrificed and the plugs and surrounding tissue were carefully dissectedand prepared for histological examination by formalin fixation. Eachplug was processed as follows; the plugs and surrounding tissues werebisected approximately through the middle into equal halves, each halfwas placed in a separate paraffin block, each block was sectioned at twolevels, offset typically by 100 μm, then each section was H&E stained,and then mounted for viewing in a microscope. At a 40×-400×magnification, functional capillaries that had been identified anddefined by the presence of red blood cells within a lumen surrounded byendothelial cells within the plugs at least a distance of 50 micronsfrom a tissue edge within the plug were counted over the entirety of theplug in each half section at two levels of each half. A count was donefor each of the two sections from each plug and the total count over allplugs from each group of five animals was tallied. The group injectedwith plugs loaded with 37 μL of 10 mM HCl solution showed no (0)capillaries. The group injected with plugs containing the 18.9 μL ofcold platelet material solution had a total of 66 functionalcapillaries. See FIGS. 1A and 1B for pictures of sections with andwithout the composition of the invention, respectively. In FIG. 1A, ascant infiltration of formation of tubes cells is seen along theperimeter of the Matrigel plug, but not throughout the volume. No fullyformed capillaries are present indicating poor angiogenic activity. InFIG. 1B, dramatic abundance of capillary and tubular forms thatinfiltrate the entire cross-section of the plug can be seen. A highermagnification view (not shown) showed an active capillary containing 10red blood cells. This image shows the powerful angiogenic properties ofthe compositions of the invention.

Example 3

The Matrigel plugs containing the heat treated product of the inventionor the acidified and urea treated product of the invention is placedsubcutaneously in mice and examined histologically after seven days.Matrigel plugs comprising acidification and urea treated products of theinvention resulted in induction of a larger number of tubes andfunctional capillaries containing red blood cells than a fraction thatwas only treated by suspension in PBS, sonication, andultracentrifugation. This effect demonstrated a dose dependence and anangiogenic response in vivo (FIGS. 2 and 3).

Example 4

A unit of expired platelets was obtained from the Bonfils blood bank andaliquoted into 50 mL centrifuge tubes which were spun at 3600 rpm for 15minutes. The supernatant fluid was pipetted from each of the pelletscarefully to avoid its disturbance. The pellets were resuspended in 14mL of 10 mM HCl in water and each solution was processed by placing theprobe of a Branson 450 sonifier into the solutions while contained in anice bucket and run for 4 minutes at duty cycle setting 20% and outputcontrol 2. The material was then loaded into high speed centrifuge tubesand centrifuged at 25,000 rpm for 45 minutes and the supernatantsolution was removed from the pellets. One portion of these solutionswas placed into a 7-8 kD MWCO dialysis bag and exhaustively dialyzedagainst 4 1 L volumes of 10 mM HCl at refrigerated temperature. Thismaterial was protein assayed using the Bradford technique and showed aconcentration of 17.4 mg/mL.

A second portion was not dialyzed but instead treated with additionalconcentrated HCl to reach a pH of 2 and this material was Bradfordassayed yielding a protein concentration of 21.0 mg/mL, suggesting thata portion of the protein material lost in the dialysis step for thefirst portion remained in this second portion.

Volumes of 3 and 10 μL of each of these materials as well as enoughadditional 10 mM HCl to make a total combined loading volume of 45 μLwere then loaded while cold into four 1300 μL portions of liquidMatrigel and injected subcutaneously on bilateral lower abdominal sitesin 3 mice to yield a total of 6 plugs for each treatment. Another equalportion of Matrigel was loaded with 45 μL of 10 mM HCl and injected assix plugs in three mice to act as a control. This dosing suggests thatapproximately 10-15% more protein material was loaded into the Matrigelplugs for the nondialyzed treatment. After seven days the mice wereeuthanized, the plugs and surrounding tissues were harvested, formalinfixed overnight, sectioned in half down the approximate midline andprepared for histological sections to be taken and made from each half.

The resulting twelve H&E stained sections (not shown) from eachtreatment and control group (3 mice, 2 plugs each, 2 halves per plug)were exhaustively viewed under a microscope at magnifications up to 400×to identify all capillaries present within the plug at least 50 micronsfrom a preexisting tissue boundary that contained red blood cells anddistinct architecture defined by an encircling endothelial cell. Thedialyzed portion gave a total of 5 and 12 capillaries for the low andhigh doses, respectively. The nondialyzed portion gave 1 and 6capillaries for the low and high doses, respectively. The control groupshowed no capillaries within the plugs.

This demonstrates an angiogenic activity in the plugs proportionate tothe dose of material loaded into the Matrigel and that the process ofdialysis led to an enhancement of this activity relative to theundialyzed portion. This also suggests that the low molecular weightprotein that was removed by dialysis has an antiangiogenic activity. Anyprocess that allows for the removal of these low molecular weighproteins, such as any type of filtration, would be expected to lead tothe same level of enhancement of angiogenic activity in the left overmaterial.

Example 5

After anesthetizing the mouse by IP injection the lower back was shaved,wiped with an antiseptic wipe, allowed to dry, and a one cmfull-thickness incision running perpendicular to the spinal axis wasmade in the skin using a sterile scissors on a pinched up portion ofskin. A small aliquot of Matrigel (25 μL) either with or without addedcomposition of the invention was placed in the wound followed by theplacement of a single suture to re-approximate the edges. Animals wereplaced in cages alone to prevent disruption of the wound by fellow cagemates. The animals were checked after one hour for any signs of adverseevents. The animals were checked daily to observe the progress of woundclosure and after 3 days an image of the wound site was collected foranalysis.

FIG. 4 shows pictures of mice treated with and without a product of theinvention. This experiment used the material obtained from patient 2described below. As can be seen, mice treated with products of theinvention showed better and/or faster wound healing compared to the micein the control group.

Matrigel Plug Method for Determining Angiogenic Activity

After anesthetizing the mouse by IP injection the lower abdomen isshaved and an aliquot of 200 μL of Matrigel, diluted by no more than 10%by the solution containing growth factors, either without (control) orwith added growth factors is placed subcutaneously in the right and leftlower abdomenal quadrants of mice. Animals were sacrificed 7 days laterfor tissue harvest. Excised plugs and surrounding tissue was fixed in10% neutral buffered formalin over night and then sectioned in half downthe middle of the plugs and subsequently processed for imbedding inparaffin. Seven (7) micron sections were stained with hematoxylin andeosin and viewed through a microscope. Counts of tubular forms andfunctional red blood cell-filled capillaries were done within the plugsfor the 10 high power fields (400×) that displayed the highest number ofthese forms over the entire plug. The area within 50 microns of the plugboundary with the native tissue was excluded from this determination toexclude vessels that were likely eprexisting before plug placement.

FIG. 2 shows counts of tubes/field within Matrigel plugs placedsubcutaneously in mice for two units of platelets obtained fromdifferent patients processed by sonication in physiologic media followedby dialysis against 10 mM HCl followed by lyophilization andresolubilization in concentrated urea and then dialysis again against 10mM HCl. The inactive material is a portion of that from patient 1 whichhas been lysed in physiologic media but not taken through any subsequentprocessing steps. The control condition involves loading the Matrigelwith vehicle without platelet growth factors, in this case 10 mM HClsolution. The doses, in nanograms/microliter were as follows: patient 1,high=67, low 22; patient 2, high=94, low=31; patient 1 inactive,high=143, low=48. FIG. 3 shows the corresponding counts ofcapillaries/field.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those skilled in the art, afterunderstanding the present disclosure. It is intended to obtain rightswhich include alternative embodiments to the extent permitted, includingalternate, interchangeable and/or equivalent structures, functions,ranges or steps to those claimed, whether or not such alternate,interchangeable and/or equivalent structures, functions, ranges or stepsare disclosed herein, and without intending to publicly dedicate anypatentable subject matter.

What is claimed is:
 1. A composition comprising a growth factor producedby a process comprising: disrupting pelletized platelets in anextraction solution to produce an insoluble material portion and theextraction solution comprising a growth factor; and removing insolublematerials and proteins having molecular weight of 2 kD or less from saidextraction solution to produce a composition comprising a growth factor.2. The composition of claim 1, wherein said method for disruptingpelletized platelets comprises sonication, homogenization, nitrogencavitation, a French pressure cell, or a combination thereof.
 3. Thecomposition of claim 1, wherein said step of removing insolublematerials and proteins comprises: (i) filtering said extraction solutionusing an ultrafiltration process to remove insoluble materials andproteins having molecular weight of 2 kD or less; or (ii) removinginsoluble materials from said extraction solution; and (a) dialyzingsaid extraction solution against an acidic solution using asemipermeable membrane having 2 kD or less MWCO; or (b) filtering saidextraction solution using an ultrafiltration process to remove proteinshaving molecular weight of 2 kD or less.
 4. The composition of claim 3,wherein said step of dialyzing comprises using a semipermeable membranehaving 8 kD or less MWCO.
 5. The composition of claim 1, wherein saidprocess further comprises subjecting said extraction solution to anultrafiltration process to remove any microorganisms that maybe present.6. The composition of claim 5, wherein said process further compriseslyophilizing said extraction solution to produce a solid compositioncomprising the growth factor.
 7. The composition of claim 6, whereinsaid process further comprises dissolving said solid composition in asolution to produce a fluid composition comprising the growth factor. 8.The composition of claim 1, wherein said growth factor comprises VEGF,TGF-β₁, TGF-β₂, PDGF-BB, EGF, or a mixture thereof.
 9. A wound healingcomposition comprising therapeutically effective amount of a compositionof claim 1 and a pharmaceutically acceptable carrier.
 10. The woundhealing composition of claim 9, wherein said pharmaceutically acceptablecarrier comprises a bandage.
 11. The wound healing composition of claim9, wherein the pharmaceutically acceptable carrier comprises apharmaceutically acceptable polymer.
 12. A method for regenerating atissue or treating a wound comprising contacting a desired site fortissue regeneration or the wound with an angiogenic compositioncomprising a composition of claim
 1. 13. The method of claim 12, whereinthe desired site for tissue regeneration comprises a burn site, a graftdonor site, an oral and maxillofacial surgery site, a mucogingivalsurgery site, a perioplastic surgery site, an oral mucosa site, or acombination thereof.
 14. A method for reducing the risk of microbialinfection in a wound comprising treating the wound with a woundtreatment composition comprising a composition of claim 1.